Ultrastructure of Spermiogenesis and Spermatozoa of Notocotylus Neyrai

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Ultrastructure of spermiogenesis and spermatozoa of Notocotylus neyrai González Castro, 1945 (Digenea, Notocotylidae), intestinal parasite of Microtus agrestis (Rodentia: Arvicolidae) in Spain

PAPA IBNOU NDIAYE1, JORDI MIQUEL1*, CARLOS FELIU1 and BERNARD MARCHAND2 1Laboratori de Parasitologia, Facultat de Farmàcia, Universitat de Barcelona, Av. Joan XXIII s/n, E-08028 Barcelona, Spain Tel. +34 (93) 402-4500; Fax: +34 (93) 402-4504; email: [email protected] 2Laboratoire Parasites et Écosystèmes Méditerranéens, Faculté des Sciences et Techniques, Université de Corse, F-20250 Corte, France

Received 24 May 2002; Accepted 18 November 2002

Summary To our knowledge, this is the first ultrastructural study on spermiogenesis and the spermatozoon of a trematode belonging to the family Notocotylidae, Notocotylus neyrai. Spermiogenesis begins with the formation of the zone of differentiation which comprises striated rootlets associated with the two centrioles and an intercentriolar body in-between. It is characterised by an asynchronic flagellar rotation and subsequent proximodistal fusion with a median cytoplasmic process. The migration of the nucleus toward the median cytoplasmic process before its fusion with the free flagella is also described. The mature spermatozoon of N. neyrai is filiform and tapered at both ends and presents all the features found in the Digenea gamete: two axonemes, mitochondrion, nucleus and two bundles of parallel cortical microtubules. Nevertheless, several characters allow us to distinguish N. neyrai from other digenetic trematodes.

Key words: Ultrastructure, spermiogenesis, spermatozoon, Notocotylus neyrai, Trematoda, Digenea, Notocotylidae

Introduction although spermatozoa have been examined in only 47 Several authors have carried out ultrastructural genera of Digenea (Chen et al., 1996; Gracenea et al., studies on Platyhelminthes for phylogenetic purposes 1997; Iomini, 1998; Tang et al., 1998; Miquel et al., (Euzet et al., 1981; Brooks, 1989; Hoberg et al., 1997; 2000; Baptista-Farias et al., 2001; Justine 2001). The Justine, 1991, 1995, 1997, 1998, 2001; Justine et al., spermatozoa described in Digenea share a few charac- 1985; Rohde, 1990; Bâ and Marchand, 1994, 1995). teristics: nucleus in posterior areas of the sperm, one or Numerous reports on the ultrastructure of the more mitochondria, parallel cortical microtubules and spermatozoon and the process of spermiogenesis are two axonemes of the 9+“1” pattern of trepaxonematan thus available: 150 papers on parasitic Platyhelminthes, Platyhelminthes (Ehlers, 1985). However, many other of which more than 80 deal with digenetic trematodes, particularities have been described, but they lack infor-

*Corresponding author. 106 P.I. Ndiaye et al. / IRD 43 (2003) 105–115 mation about the pattern of spermiogenesis and undergo a rotation, become parallel to the cytoplasmic spermatozoon particularities for determine phylo- extension and fuse with it (Figs. 2–4). The rotation and genetic purposes. proximodistal fusion of the free flagella are asyn- After the review of Simón-Vicente et al. (1985), chronous: one flagellum fuses before the other (Fig. 5). three species of the genus Notocotylus are accepted as Electron-dense areas between the dorsal and ventral parasites of rodents in Europe: Notocotylus noyeri cortical microtubules are observed in the median Joyeux, 1922, N. neyrai González Castro, 1945 and cytoplasmic process before the proximodistal fusion of N. gonzalezi Simón Vicente, Mas Coma, López the free flagella (Fig. 5). They are the origin of the Román, Tenora et Gállego, 1985. There are limited future attachment zones (Fig. 6). The fusion of the ultrastructural data available on gametogenesis in the flagella with the median cytoplasmic process deter- Notocotylidae family. Here, we present the first mines the appearance of two sets of cortical micro- ultrastructural study on the spermiogenesis and tubules (Figs. 5, 6). The nucleus migrates toward the spermatozoon of one species of this family, N. neyrai. median cytoplasmic process before the proximodistal fusion (Figs. 3, 4). However, a longitudinal section of the differentiation zone revealed that nucleus migration Materials and Methods takes place after flagellar rotation (Figs. 3–5). The mitochondria migrate along the spermatid body after Live N. neyrai specimens were collected from the complete fusion of the two flagella and the median Microtus agrestis from Vall d’Arties (Lleida, Spain). cytoplasmic process (Figs. 6–8). Finally, the ring of Adults were kept in 0.9% NaCl solution. Several arched membranes is strangled and the young sper- portions of these specimens were dissected and fixed in matozoon detaches from the residual cytoplasm. cold (4°C) 2.5% glutaraldehyde in 0.1 M sodium The mature spermatozoon of N. neyrai (Figs. 9–26, cacodylate buffer at pH 7.2 for 2 h, rinsed in 0.1 M 28I–VI) is characterised by the presence of two sodium cacodylate buffer at pH 7.2, postfixed in cold axonemes, at least two mitochondria, nucleus and two (4°C) 1% osmium tetroxide in the same buffer for 1 h, sets of parallel cortical microtubules. Other ultra- rinsed in 0.1 M sodium cacodylate buffer at pH 7.2, structural features and their location are described dehydrated in ethanol solutions and propylene oxide, below. The observation of a large number of embedded in Spurr and polymerised at 60ºC for 48 h. longitudinal and transverse sections allowed us to Ultrathin sections (60–90 nm) of testes and seminal establish six regions with distinct ultrastructural ducts were obtained using a Reichert-Jung Ultracut E features: ultramicrotome, placed on 200 mesh copper grids and double-stained with uranyl acetate and lead citrate following Reynolds (1963). Copper grids were examined under a Hitachi H-600 Figs. 1–6. Spermiogenesis of Notocotylus neyrai. Fig. 1. Zone EM at 75 kV in the “Serveis Cientificotècnics” of the of differentiation showing the growth of the first flagellum. Ib, University of Barcelona (Spain) and in the University intercentriolar body; N, nucleus. Bar = 0.5 µm. Fig. 2. Two of Corsica (Corte, France). cross sections of zones of differentiation. One of them shows the two centrioles, the intercentriolar body (Ib) and the nucleus (N). The other shows the median cytoplasmic Results process (Mcp) and the two free flagella before the proxi- Spermiogenesis in N. neyrai (Figs. 1–8, 27A–F) modistal fusion. Bar = 0.5 µm. Fig. 3. Longitudinal section of a zone of differentiation showing the nuclear migration. begins with the formation of a differentiation zone in Am, arched membranes; N, nucleus; Sr, striated roots. Bar = the spermatid. This is a conical area, characterised by 1 µm. Fig. 4. Longitudinal section of a zone of differen- the presence of arched membranes and bordered by a tiation after the flagellar rotation of the two flagella. Am, layer of cortical microtubules (Fig. 1). It also contains arched membranes; Ib, intercentriolar body; Mcp, median two centrioles separated by an intercentriolar body, cytoplasmic process; N, nucleus; Sr, striated roots. Bar = which show an associated striated rootlet (Figs. 1–4) 1 µm. Fig. 5. Cross section of a zone of differentiation after and develop a flagellum that grows externally, and an the proximodistal fusion of the first flagellum. Note the appearance of dense material in the zone of fusion (arrow emerging median cytoplasmic process (Figs. 1, 3, 4). heads). C1, centriole of the first flagellum; F2, second The cortical microtubules initiate their migration along flagellum; Mcp, median cytoplasmic process. Bar = 0.5 µm. this median process (Fig. 2). At the beginning of their Fig. 6. Cross section of spermatid showing the attachment development, the two flagella grow orthogonal to the zones (arrow heads) and the mitochondrion (Mt). Bar = median cytoplasmic process (Fig. 1). Thereafter, they 0.5 µm. P.I. Ndiaye et al. / IRD 43 (2003) 105–115 107 108 P.I. Ndiaye et al. / IRD 43 (2003) 105–115

Figs. 7, 8. Spermiogenesis of Notocotylus neyrai. Fig. 7. Longitudinal sections of two zones of differentiation. The mitochondrion (Mt) starts its migration. Am, arched membranes; N, nucleus. Bar = 0.5 µm. Fig. 8. Longitudinal sections of spermatids showing the mitochondrial migration in the final stages of spermiogenesis. Striated roots (Sr) remain at the base of the spermatid and dense material (arrow head) appears before the strangulation of the ring of arched membranes (Am). Mt, mitochondrion. Bar = 1 µm. P.I. Ndiaye et al. / IRD 43 (2003) 105–115 109

Region I (Figs. 9–17, 28I). This region encompases the spermatozoon exhibits only several axonemal micro- anterior end of the spermatozoon. It is characterised by tubules (Figs. 22, 26). the presence of two axonemes of the 9+“1” pattern of Trepaxonemata, one mitochondrion and external orna- Discussion mentation of the plasma membrane. At the beginning of the process, it is sharp (Fig. 9), shows a single Spermiogenesis in N. neyrai follows the general axoneme (Figs. 9, 10, 13) and lacks cortical micro- pattern found in all the digenetic trematodes studied to tubules (Figs. 9, 10). A second axoneme, cortical date (Burton, 1972; Erwin and Halton, 1983; Gracenea microtubules and external ornamentation soon appear et al., 1997; Miquel et al., 2000; Baptista-Farias et al., (Figs. 11–17). Cortical microtubules are numerous 2001, etc.). In Opecoeloides furcatus, Miquel et al. (30–40), parallel to the long axis of the spermatozoon, (2000) described an asynchronic process of proximo- and they appear as a continuous submembranous layer. distal fusion and the first fused flagellum migrates to The plasma membrane is devoid of submembranous distal areas of the spermatid after its fusion with the cortical microtubules only in a small area where two median cytoplasmic process but before the complete attach points are observed (Figs. 12, 16). Most trans- proximodistal fusion of the second flagellum. This may verse sections of region I reveal a mitochondrion account for the origin of the anterior extremity of the (Figs. 16, 17) and some of them show spine-like bodies future spermatozoa with a single axoneme. In the (Figs. 12, 15, 17). These structures consist of triangular anterior extremity of the N. neyrai spermatozoon, we prominences containing a submembranous and also observed a single axoneme both in cross and electron-dense spherical vesicle. longitudinal sections as a result of the relative displace- Region II (Figs. 18–20, 28II). This region is charac- ment of one of the axonemes with respect to the other. terised by the disappearance of membranous ornamen- The present study and most published reports tation, the absence of mitochondria and a clear bilateral (Burton, 1972; Erwin and Halton, 1983; Hendow and symmetry (Figs. 18, 20). Cross sections of the sperma- James, 1988; Iomini and Justine, 1997; Miquel et al., tozoon in this region show only two axonemes and two 2000; Baptista-Farias et al., 2001) describe the nucleus bundles of cortical microtubules (Figs. 18, 20). migration toward the median cytoplasmic process Granules of glycogen appear progressively between the before the migration of the mitochondrion, in contrast axonemes (Figs. 18–20). with the results obtained by Gracenea et al. (1997) in Region III (Figs. 20, 21, 24, 28III). In addition to Postorchigenes gymnesicus. Only these authors have the structures observed in Region II, this region described mitochondrion migration before the nucleus presents a mitochondrion between the two axonemes migration in Digenea. (Figs. 20, 21, 24), which may differ from that observed The basal bodies, striated rootlets and intercen- in Region I. Therefore, we show a schematic drawing triolar body have never been described in the mature of sperm containing two mitochondria (Fig. 28I–VI). spermatozoon. Both in trematodes and cestodes, it is Region IV (Figs. 20, 26, 28IV). This region is thus assumed that these structures remain in the characterised by the simultaneous presence of two residual cytoplasm and degenerate (Burton, 1972; axonemes, mitochondrion, nucleus and abundant Mokhtar-Maamouri and Swiderski, 1975; Rees, 1979; glycogen granules (Figs. 20, 26). In a transversal Erwin and Halton, 1983; Brunanska et al., 2001). In section, the cortical microtubules form two bundles late N. neyrai spermiogenesis, a crescent proximity of (Figs. 20, 26). striated roots to the arched membranes is noted, and Region V (Figs. 20, 22, 23, 28V). This region this fact support this hypothesis. presents a single axoneme, mitochondrion, nucleus and The presence of more than one mitochondrion has numerous glycogen granules (Figs. 20, 22). The cross been described in most digeneans, namely Haemato- sections of the nucleus are larger than in region IV. loechus medioplexus (Burton, 1972), Pharyngosto- Region VI (Figs. 22, 24–26, 28VI). This region moides procyonis (Grant et al., 1976), Cryptocotyle includes the posterior part of the mature spermatozoon. lingua (Rees, 1979), Paragonimus ohirai (Orido, The cross sections reveal a single axoneme, nucleus 1988), P. gymnesicus (Gracenea et al., 1997) and and glycogen granules (Figs. 22, 24, 25). The disrup- N. neyrai (present study). However, the presence of a tion of the axoneme occasionally begins at the nucleus single mitochondrion has been reported in other (Fig. 25). Cortical microtubules, glycogen granules and species of digeneans such as Bucephaloides graci- nucleus disappear before the axonemal doublets and lescens (Erwin and Halton, 1983), Echinostoma singlets. Therefore, the posterior extremity of the caproni (Iomini and Justine, 1997) and O. furcatus Figs. 9–18. Spermatozoon of Notocotylus neyrai. Fig. 9. Longitudinal section of the anterior spermatozoon extremity (Ase). Bar = 0.5 µm. Fig. 10. Cross section of the anterior area of Region I characterised by the presence of a single axoneme. Bar = 0.5 µm. Fig. 11. Cross section of Region I showing the external membranar ornamentation (Eo) covering only one of the two axonemes. Bar = 0.5 µm. Fig. 12. Cross section of Region I showing the spine-like body (Sb) and the presence of only two attachment zones (arrow heads). Bar = 0.5 µm. Fig. 13. Longitudinal section of Region I at the level of the beginning of the extramembranar ornamentation (arrow head) and showing the appearance of the second axoneme (arrow). Bar = 0.5 µm. Fig. 14. Longitudinal section of Region I showing the external ornamentation of the plasma membrane (Eo). Bar = 0.5 µm. Fig. 15. Tangent section of Region I showing the spine-like body (Sb). Bar = 0.5 µm. Fig. 16. Cross section of Region I showing the mitochondrion (Mt). Bar = 0.5 µm. Fig. 17. Another cross section of Region I in area with simultaneous presence of mitochondrion (Mt) and spine-like body (Sb). Eo, extramembranar ornamentation. Bar = 0.5 µm. Fig. 18. Several cross sections of Region II. Note the presence of four attachment zones (arrow heads) in each section. Bar = 0.5 µm. P.I. Ndiaye et al. / IRD 43 (2003) 105–115 111

Figs. 19–26. Spermatozoon of Notocotylus neyrai. All bars = 0.5 µm. Fig. 19. Longitudinal section of Region II. Fig. 20. Cross sections of Regions II, III, IV and V. In Region V note the two attachment zones (arrow heads) G, granules of glycogen; Mt, mitochondrion. Fig. 21. Longitudinal section of Region III. Mt, mitochondrion. Fig. 22. Cross sections of Regions V and VI. D, doublet; Mt, mitochondrion; N, nucleus. Fig. 23. Longitudinal section of Region V. G, granules of glycogen; Mt, mitochondrion; N, nucleus. Fig. 24. Cross sections of Regions III and VI. Cm, cortical microtubules; N, nucleus. Fig. 25. Cross section of Region VI showing the axonemal disorganization at the level of nucleus. D, doublets. Fig. 26. Cross sections of Regions IV and VI. Mt, mitochondrion; N, nucleus; S, singlets. 112 P.I. Ndiaye et al. / IRD 43 (2003) 105–115

Fig. 27. (A–F). Diagram showing the main stages of spermiogenesis of Notocotylus neyrai.

(Miquel et al., 2000). Burton (1972) has suggested that originates from fusion of the three processes and has numerous mitochondria of the spermatid accompany no ornamentation. the nucleus into the median cytoplasmic process where Until today, spine-like bodies have previously been they apparently fuse together to form the long mito- described only in Opecoeloides furcatus (Miquel et al., chondrion of the mature sperm. Regarding parasitic 2000), and in this species they are located at the level Platyhelminthes, a single mitochondrion has also been of mitochondrial areas. These structures probably may described in the spermatozoon of monogeneans but, to be formed in late spermiogenesis. our knowledge, not in cestodes (Justine, 1995, 1998, Attachment zones in mature spermatozoa indicate 2001). the area of fusion of flagella with the median cyto- Most studies reveal the presence of an external plasmic process (Burton, 1972). In anterior and ornamentation of the plasma membrane in the sper- posterior areas of mature spermatozoon of N. neyrai, matozoon of digeneans (Jamieson and Daddow, 1982; only two attachment zones are detected, pointing to the Justine and Mattei, 1982a, 1982b; Gracenea et al., origin of the anterior areas of the first axoneme present 1997; Iomini and Justine, 1997; Miquel et al., 2000), in the anterior extremity of the spermatozoon from the but the localisation of this ornamentation along the zone of differentiation. This flagellum rotates, but spermatozoon can vary in these species. Structures proximodistal fusion does not occur in its anterior similar to external ornamentation of the plasma extremity. In contrast, the second axoneme fuses totally membrane have also been described in the anterior part with the median cytoplasmic process during the proxi- of the spermatozoon of a few monogeneans: Microtyle modistal fusion and determines the appearance of two sp. (Microcotylidae) and Pseudomazocraes cf. monsi- attachment zones throughout its length. These events vaisae (Chauhaneidae) (Justine and Mattei, 1985). are confirmed by the presence of a continuous layer of According to Justine (1991, 1995), this external orna- submembranous cortical microtubules not organised in mentation characterises the region of the spermatozoon ventral and dorsal bundles. In Echinostoma caproni, originating from the zone of differentiation, which similar sections (containing two axonemes, extramem- corresponds with the anterior areas in the mature branar ornamentation and mitochondrion) but with four spermatozoon, whereas the rest of the spermatozoon attachment points, as a result of fusion of the two free P.I. Ndiaye et al. / IRD 43 (2003) 105–115 113

Fig. 28. (I–VI). Diagram showing the ultrastructural organization of mature sperm of Notocotylus neyrai. To make the diagram clearer, the granules of glycogen are not included in the longitudinal sections of the drawing. 114 P.I. Ndiaye et al. / IRD 43 (2003) 105–115 flagella with the median cytoplasmic process during Cooperación Internacional –AECI of the Ministerio de spermiogenesis, have been described (Iomini and Asuntos Exteriores. Justine, 1997). On the other hand, the posterior spermatozoon areas of N. neyrai with a single axoneme References also show only two attachment zones as a result of the fusion of the second flagellum with the median cyto- Bâ, C.T. and Marchand, B., Similitude ultrastructurale des plasmic process and reveal the different growth in spermatozoïdes de quelques Cyclophyllidea. Parasite, 1 (1994) 51–55. length of the free flagella before their proximodistal Bâ, C.T. and Marchand, B., Spermiogenesis, spermatozoa fusion. and phyletic affinities in the Cestoda. 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